The present invention relates to a thermostable glucoamylase suitable for, e.g., starch conversion, e.g., for producing glucose from starch. The present invention also relates to the use of said thermostable glucoamylase in various processes, in particular in the saccharification step in starch convention processes.
Glucoamylases (1,4-xcex1-D-glucan glucohydrolase, EC 3.2.1.3) are enzymes which catalyze the release of D-glucose from the non-reducing ends of starch or related oligo- and polysaccharide molecules.
Glucoamylases are produced by several filamentous fungi and yeasts, including Aspergillus niger and Aspergillus awamori. 
Commercially, the glucoamylases are used to convert corn starch which is already partially hydrolyzed by an xcex1-amylase to glucose. The glucose may further be converted by glucose isomerase to a mixture composed almost equally of glucose and fructose. This mixture, or the mixture further enriched with fructose, is the commonly used high fructose corn syrup commercialized throughout the world. This syrup is the world""s largest tonnage product produced by an enzymatic process. The three enzymes involved in the conversion of starch to fructose are among the most important industrial enzymes produced.
One of the main problems existing with regard to the commercial use of glucoamylase in the production of high fructose corn syrup is the relatively low thermal stability of glucoamylases, such as the commercially available Aspergillus niger glucoamylase (i.e., (sold as AMG by Novo Nordisk A/S). The commercial Aspergillus glucoamylase is not as thermally stable as xcex1-amylase or glucose isomerase and it is most active and stable at lower pH""s than either xcex1-amylase or glucose isomerase. Accordingly, it must be used in a separate vessel at a lower temperature and pH.
U.S. Pat. No. 4,247,637 describes a thermostable glucoamylase having a molecular weight of about 31,000 Da derived from Talaromyces duponti suitable for saccharifying a liquefied starch solution to a syrup. The glucoamylase is stated to retain at least about 90% of its initial glucoamylase activity when held at 70xc2x0 C. for 10 minutes at pH 4.5.
U.S. Pat. No. 4,587,215 discloses a thermostable amyloglucosidase derived from the species Talaromyces thermophilus with a molecular weight of about 45,000 Da. The disclosed amyloglucosidase (or glucoamylase) loses its enzymatic activity in two distinct phases, an initial period of rapid decay followed by a period of slow decay. At 70xc2x0 C. (pH=5.0) the half-life for the fast decay is about 18 minutes with no measurable loss of activity within an hour in the second phase of decay.
Bunni L et al., (1989), Enzyme Microb. Technol., Vol. 11, p. 370-375. concerns production, isolation and partial characterization of an extracellular amylolytic system composed of at least one form of xcex1-amylase and one form of an xcex1-glucosidase produced by Talaromyces emersonii CBS 814.70. Only the xcex1-amylase is isolated, purified and characterized.
The present invention is based upon the finding of a novel thermostable glucoamylase suitable for use, e.g., in the saccharification step in starch conversion processes.
The terms xe2x80x9cglucoamylasexe2x80x9d and xe2x80x9cAMGxe2x80x9d are used interchangeably below.
The thermal stability of the glucoamylase of the invention is measured as T1/2 (half-life) using the method described in the xe2x80x9cMaterials and Methodsxe2x80x9d section below.
The inventors of the present invention have isolated, purified and characterized a thermostable glucoamylase from a strain of Talaromyces emersonii now deposited with the Centraalbureau voor Schimmelcultures under the number CBS 793.97.
When applied to a protein, the term xe2x80x9cisolatedxe2x80x9d indicates that the protein is found in a condition other than its native environment. In a preferred form, the isolated protein is substantially free of other proteins, particularly other homologous proteins (i.e., xe2x80x9chomologous impuritiesxe2x80x9d (see below)).
It is preferred to provide the protein in a greater than 40% pure form, more preferably greater than 60% pure form. Even more preferably it is preferred to provide the protein in a highly purified form, i.e., greater than 80% pure, more preferably greater than 95% pure, and even more preferably greater than 99% pure, as determined by SDS-PAGE.
The term xe2x80x9cisolated enzymexe2x80x9d may alternatively be termed xe2x80x9cpurified enzymexe2x80x9d.
The term xe2x80x9chomologous impuritiesxe2x80x9d means any impurity (e.g. another polypeptide than the polypeptide of the invention) which originates from the homologous cell, from where the polypeptide of the invention is originally obtained.
The isolated glucoamylase has a very high thermal stability in comparison to prior art glucoamylases, such as the Aspergillus niger glucoamylase (available from Novo Nordisk A/S under the trade name AMG). The Txc2xd (half-life) was determined to be about 120 minutes at 70xc2x0 C. (pH 4.5) as described in Example 2 below. The Txc2xd of the recombinant T. emersonii AMG expressed in yeast was determined to be about 110 minutes as described in Example 12.
Therefore, in the first aspect the present invention relates to an isolated enzyme with glucoamylase activity having a T1/2 (half-life) of at least 100 minutes in 50 mM NaOAc, 0.2 AGU/ml, pH 4.5, at 70xc2x0 C.
In the second aspect the invention relates to an enzyme with glucoamylase activity comprising one or more of the partial sequences shown in SEQ ID Nos. 1-6 or the full length enzyme shown in SEQ ID NO: 7 or an enzyme with glucoamylase activity being substantially homologous thereto.
The term xe2x80x9cpartial sequencexe2x80x9d denotes a partial polypeptide sequence which is comprised in a longer polypeptide sequence, wherein said longer polypeptide sequence is having the activity of interest.
The invention also relates to the cloned DNA sequence encoding the glucoamylase of the invention.
Further, the invention also relates to a process of converting starch or partially hydrolyzed starch into a syrup containing, e.g., dextrose, said process including the step of saccharifying starch hydrolyzate in the presence of a glucoamylase of the invention.
It is an object of the invention to provide a method of saccharifying a liquefied starch solution, wherein an enzymatic saccharification is carried out using a glucoamylase of the invention.
Furthermore, the invention relates to the use of a glucoamylase of the invention in a starch conversion process, such as a continuous starch conversion process. In an embodiment of the continuous starch conversion process it includes a continuous saccharification step.
The glucoamylase of the invention may also be used in processes for producing oligosaccharides or specialty syrups.
Finally, the invention relates to an isolated pure culture of the microorganism Talaromyces emersonii CBS 793.97 or a mutant thereof capable of producing a glucoamylase of the invention.